Exoenzyme S (ExoS) is an ADP-ribosylating toxin produced by the opportunistic pathogen Pseudomonas aeruginosa which requires direct contact between the bacterium for translocation into the host cell. Once within the target cell, ExoS causes complex effects on cellular processes resulting in inhibition of DNA synthesis and alterations in the cytoskeleton affecting cell structure, movement, microvilli and focal adhesion turnover. Studies from the PI's laboratory have led to the development of a bacterial-eukaryotic cell co-culture system which allowed the toxic effects of ExoS to be identified. This system was also used to provide insight into the cellular mechanism of action of ExoS by identifying Ras as an in vivo target of ExoS ADP-ribosyltransferase (ADPRT) activity. The complex effects of ExoS on cellular function can to some extent be explained by its multi-domain structure. Current data support the possibility that ExoS can cause transient alterations in cytoskeletal structure via non-ADPRT mechanism which is then coordinated with the ADP-ribosylation of specific cellular proteins leading to long-term alterations in cytoskeletal structure and inhibition of DNA synthesis. The cellular mechanism for the diverse affects of ExoS on cell function, however, remains unknown, and may relate to the ADP-ribosylation of cellular Ras, which plays an integral role in multiple signal transduction pathways, the ADP-ribosylation of other cellular proteins, or to indirect effects of ExoS on proteins linked to the cytoskeleton. The goal of this proposal is to identify cellular processes and signaling pathways affected by ExoS following bacterial translocation. The purpose of the first specific aim is to gain further understanding of cellular proteins directly affected by ExoS by examining the in vivo substrate specificity of ExoS ADPRT activity. The second aim focuses on Ras, examining how ADP-ribosylation of Ras by ExoS affects Ras mediated cell signaling events. The third aim examines the cellular mechanism of ExoS associated alteration in cytoskeletal structure, focusing on both enzymatic and non-enzymatic effects of ExoS on Rho, Rac, and Cdc42, which function in the regulation of cytoskeletal structure, the combined picture will provide insight into how ExoS influences cellular signaling, and in turn, the role of ExoS in Pseudomonas pathogenesis.